Throughout this application, various references are cited to describe more fully the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.
Hepatocellular carcinoma (HCC) is the most common solid-organ tumor in the world, being responsible for more than 1 million deaths annually (Parkin, et al., (1999) C A. Cancer J. Clin., 49:33-64, and Okuda, et al., (1993), Neoplasms of the Liver in Diseases of the Liver, 7th edition 1236).
The incidence of HCC is increasing in most Western countries (Deuffic, et al., (1998), Lancet 351:214-215). Although good epidemiological studies are not available, data from Health Canada indicate that the death rate from HCC in men has nearly doubled over the last 15 years. The demographics of patients with viral hepatitis suggest that the incidence will double again in the next 10 years (Zou, et al., (2000), Can. J. Gastroenterol., 14:575-580).
HCC is usually asymptomatic at early stages and has a great propensity for intravascular or intrabiliary invasion, even when the primary tumor is small (Fong, et al., (2001), Cancer of the Liver and Biliary Tree. in Cancer: Principles & Practice of Oncology, 6th Edition, 1162-1199). As a result, HCC is generally at an advanced stage when discovered. Historically, only 10-20% of primary HCCs are found to be resectable at the time of diagnosis (Fong, et al., (2001), Cancer of the Liver and Biliary Tree in Cancer: Principles & Practice of Oncology, 1162-1199). More recently, with the advent of HCC screening programs, the proportion of potentially curable tumors is increasing, although most patients still have incurable disease at the time of diagnosis.
HCC is associated with chronic liver injury, primarily chronic viral hepatitis and alcoholic liver disease (Rustgi, (1987), Gastroenterol. Clin. North Am., 16: 545-551). The highest incidence of HCC is found in areas were hepatitis B virus (HBV), and hepatitis C virus (HCV) are endemic. In the case of HBV, it has been demonstrated that the relative risk of developing HCC is 50- to 100-fold greater in individuals with evidence of chronic infection than in non-infected individuals (Beasley, et al., (1994), Epidemiology of hepatocellular carcinoma in Viral hepatitis and liver disease, 209). Unlike other causes of HCC, chronic hepatitis B cirrhosis is not a necessary precondition for the development of HCC. There are no estimates of relative risk with chronic HCV, but the incidence of HCC in cirrhotic carriers of HCV may be as high as 5% per year, compared to 0.5% for HBV carriers (Di Bisceglie, (1995), Semin. Liver Dis., 15:64-69). Persistent HCV infection is the cause of 70% of the cases of HCC in Japan, and the most likely reason for the rising incidence of HCC in North America is the increased spread of HCV infection in the population (Hasan, et al., (1990), Hepatology, 12:589-591 and El-Serag, et al., (1999), N. Engl. J. Med., 340:745-750).
Several chemicals have been linked to the development of HCC. The most important of them is ethanol, and alcohol abuse has been linked to HCC (Schiff, (1997), Hepatology, 26:39S-42S and Nalpas, et al., (1995), Alcohol, 12:17-120). Ethanol is thought to produce HCC through the generation of hepatic cirrhosis or as a co-carcinogen with other agents such as HBV and HCV. Aflatoxins produced by several fungi have also been linked to HCC (Yu, (1995), J. Gastroenterol. Hepatol., 10:674-682). These fungi tend to grow on grains, peanuts, and other food products, and are the most frequent cause of food spoilage.
The diagnosis of HCC is relatively straightforward in patients with a space-occupying lesion on ultrasonography or computed tomography (CT), and serum alphafetoprotein (AFP) of more than 500 ng/ml (Fong, et al., (2001), in Cancer of the Liver and Biliary Tree in Cancer: Principles & Practice of Oncology, 6th Edition, 1162-1199). In general, however, by the time these conditions are met, the HCC is untreatable, since very frequently the AFP is not diagnostically elevated.
Diagnosis by imaging of small lesions is relatively inaccurate, whether by ultrasonography, CT scanning or MRI (Fong, et al., (2001), in Cancer of the Liver and Biliary Tree in Cancer: Principles & Practice of Oncology, 6th Edition, 1162-1199); and Murakami, et al., (1995), Detectability of hypervascular hepatocellular carcinoma by arterial phase images of MR and spiral CT. Acta Radiol., 36: 372-376). In particular, the two lesions which may mimic HCC radiologically are cirrhotic nodules and dysplastic nodules. Liver biopsy of small lesions is also insufficiently sensitive or specific (Levy, et al., (2001), Ann. Surg., 234:206-209). Even with a needle biopsy, a well-differentiated cancer may be difficult to distinguish from benign lesions due to the limited amount of material usually obtained by this procedure (Fong, et al., (2001), in Cancer of the Liver and Biliary Tree in Cancer: Principles & Practice of Oncology 6th Edition, 1162-1199). Thus despite the advances in imaging technology, there is still a need for a suitable molecular marker to distinguish HCC from benign liver lesions in difficult cases.
The size of a tumor is a significant risk factor for intrahepatic spread and metastasis of HCC (Yuki, et al., (1990), Cancer, 66:2174-2179). In addition, many more treatment options are available for patients with small tumors. Symptomatic tumors are generally large, and beyond therapeutic intervention. Another situation where a sensitive and specific marker for HCC would be useful is in screening of patients at risk, such as chronic carriers of HBV and individuals with cirrhotic HCV. Although this screening is widely applied, there are as yet few data to suggest that it is effective in reducing disease (Collier, et al., (1998), Hepatology, 27:273-278). One of the reasons why screening has not been shown to be effective is that the serological test currently used, namely sequential AFP assays, has low sensitivity and specificity (Collier, et al., (1998), Viral Hepatitis Reviews, 4:31-41). Thus, improved test systems are needed to improve screening for HCC.
The only molecular marker that has been widely used for the screening and diagnosis of HCC is alphafetoprotein (AFP). This protein is synthesized in large quantities during embryonic development by the yolk sac and by the liver (Chan, et al., (1999), in Tumor Markers. in Tietz textbook of clinical chemistry, 3rd, 722-749, Taketa, K (1990), Hepatology, 12:1420-1432). AFP concentration decreases gradually after birth to <10 ng/ml in 12-18 months. AFP reappears in maternal serum during pregnancy. Increased circulating AFP has been associated with HCC, gastric carcinoma, lung cancer, pancreatic cancer, biliary tract cancer, and testicular carcinoma (Chan, et al., (1999), in Tumor Markers. in Tietz textbook of clinical chemistry, 3rd, 722-749).
If AFP levels of 20 ng/ml or higher are considered diagnostic, 60 to 80% of HCC cases are detected but, in the case of small tumors, sensitivity is significantly lower (40%) (Trevisani, et al., (2001), J. Hepatol., 34:570-575). Another problem with the use of AFP as a marker for HCC is its lack of specificity; significant increases of AFP (20-200 ng/ml) are seen in a considerable number of patients with chronic liver diseases (Collier, et al., (1998), Viral Hepatitis Reviews, 4:31-41). It has been reported that 15-58% of patients with chronic hepatitis, and 11-47% with cirrhosis had increased serum AFP (Taketa, (1990), Hepatology, 12:1420-1432). It is, therefore, not uncommon that serum AFP levels in patients with HCC and cirrhosis overlap, which confounds the interpretation of the results of the AFP assay. Consequently, the usefulness of AFP measurement as a surveillance tool for patients at risk for HCC has been questioned (Sherman, (2001), J. Hepatol., 34:603-605). It has been proposed, therefore, that the only circumstance in which the measurement of AFP is justified is for the confirmation of an initial diagnosis based on an imaging technique (Sherman, (2001), J. Hepatol., 34:603-605).
In view of the unreliability of AFP levels, most screening regimens include ultrasonography, which is highly sensitive to liver masses. However, ultrasonography lacks specificity, and cannot reliably distinguish between HCC, cirrhotic nodule and dysplastic nodule when the lesions are smaller than about 2 cm.
In the last few years, several new potential markers for HCC have been investigated, but nothing clearly superior to AFP has been found (Seow, et al., (2001), Proteomics, 1:1249-1263). For example, des-gamma-carboxy prothrombin has been proposed as a potential marker. However, this molecule, like AFP, is not useful for the detection of small tumors (Nomura, et al., (1999), Am. J. Gastroenterol., 94:650-654).
In 1997, Hsu et al., reported that, by performing differential mRNA display analysis of normal liver and HCC, they had identified a transcript that was upregulated in HCC (Hsu, et al., (1997), Cancer Res., 57:5179-5184). This transcript, which they named MXR7, turned out to be Glypican-3 (GPC3). GPC3 is a heparan sulfate proteoglycan that is bound to the cell surface by a lipid tail (Duenas Gonzales, et al., (1998), J. Cell. Biol., 141:1407-1414). Hsu et al. found that GPC3 mRNA was expressed in 143 out of 191 (74.8%) primary and recurrent HCCs, but in only 5 of 154 (3.2%) normal livers.
A second study found over-expression of GPC3 mRNA in 75% of HCC cases, but no over-expression was detected in focal nodular hyperplasia and cirrhotic liver (Zhu et al., (2001), Gut, 48, 558-564).
These studies analyzed only mRNA levels and it is known that mRNA levels do not always correlate with protein expression and secretion. In addition, only a limited number of liver proteins are normally secreted to the circulation.
While these results suggested that GPC3 mRNA analysis in liver tissue might be useful for the detection of HCC, this type of analysis is invasive, since it requires the isolation of tumor tissue. Furthermore, mRNA analysis is time-consuming and difficult to perform routinely.
There is therefore a need for a serum marker which can be used for screening (i.e. which will be positive in a high proportion of asymptomatic patients with small HCC's). In patients where an ultrasound has identified a small mass lesion, the test should also be able to reliably separate patients with HCC from those with non-malignant lesions.
In conclusion, there is still a need for better molecular markers for HCC, particularly for the surveillance of high risk populations, such as HCV patients with established cirrhosis. The search for such markers has been very difficult due to the high degree of heterogeneity that characterizes HCCs (Thorgeirsson, et al., (2002), Nature Genet, 31:339-346).